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The Journal of Neuroscience, February 15, 2001, 21(4):1127-1136
Structural Determinants of Fast Desensitization and
Desensitization-Deactivation Coupling in GABAA
Receptors
Matt T.
Bianchi1,
Kevin
F.
Haas1, and
Robert L.
Macdonald2, 3
1 Neuroscience Graduate Program and the Departments of
2 Neurology and 3 Physiology, University of
Michigan, Ann Arbor, Michigan 48104-1687
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ABSTRACT |
Fast IPSCs in the brain are predominantly caused by
presynaptic release of GABA that activates GABAA
receptor (GABAAR) channels. The IPSCs are shaped by the
gating and desensitization properties of postsynaptic
GABAARs. Specifically, fast desensitization has been
suggested to decrease IPSC amplitude and to increase IPSC duration by
slowing deactivation; however, the mechanisms underlying desensitization, deactivation, and their coupling are poorly
understood. Consistent with this suggestion, 
1
3
2L
GABAARs desensitize with a prominent fast phase and
deactivate slowly, whereas 13
GABAARs desensitize
without a fast phase and deactivate rapidly. Using the
concentration-jump technique applied to excised patches, we studied
GABAARs containing chimeras or exchange mutants between and 2L subunits to gain insight into the structural bases for fast
desensitization and its coupling to deactivation. We demonstrated that
the N terminus and two adjacent residues (V233, Y234) in the first
transmembrane domain (TM1) of the subunit were both required to
abolish fast desensitization. Additionally, these residues in TM1 of
the 2L subunit (Y235, F236) were critical for desensitized states to
prolong deactivation after removal of GABA, because mutations resulted
in accelerated deactivation despite unaltered desensitization time
course. Interestingly, control of desensitization and deactivation was
independent of the identity (2L or subunit sequence) of TM2,
indicating that structures related to the putative channel gate may
play a less direct role in desensitization than previously suggested.
Key words:
GABAA receptor; desensitization; deactivation; rapid kinetics; concentration-jump; recombinant
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INTRODUCTION |
Fast synaptic inhibition in the
brain is predominantly attributable to release of GABA, which activates
GABAA receptor (GABAAR) channels. GABAARs are members of a superfamily of
ligand-gated ion channels (Ortells and Lunt, 1995
), and seven different
mammalian subunit families (, , , , 
, 
, 
) and
their subtypes (1-6, 1-3, 1-3) have been reported
(Macdonald and Olsen, 1994; Davies et al., 1997; Hedblom and Kirkness,
1997; Bonnert et al., 1999). Multiple GABAAR
isoforms, the majority of which are and heteromers
(McKernan and Whiting, 1996), are composed of different combinations of
five subunits (Nayeem et al., 1994) that together form a transmembrane
chloride ion channel.
GABA concentration at central synaptic clefts has been estimated to
reach 500-1000 µM rapidly (<1 msec) (Maconochie et al., 1994; Jones and Westbrook, 1995) before decaying within milliseconds because of diffusion and presynaptic terminal reuptake (Clements, 1996). The current relaxation time of IPSCs after clearance of GABA
(deactivation), however, is substantially longer than predicted by the
relatively low affinity and short burst durations of
GABAARs (Macdonald et al., 1989; Fisher and
Macdonald, 1997). Because the time course of IPSCs can be reproduced
reasonably by native and recombinant GABAARs in
excised membrane patches with 1-10 msec pulses of saturating GABA
concentrations (Jones and Westbrook, 1995; Tia et al., 1996; Galarreta
and Hestrin, 1997; Haas and Macdonald, 1999), its long duration
relative to the brief GABA transient is likely attributable to
intrinsic channel kinetics that do not depend on (although they may be
modified by) a neuronal milieu.
Continued application of GABA to GABAARs leads to
a decline in current, termed desensitization, that occurs with fast
(~10 msec), intermediate (~150 msec), and slow (~1500 msec) rates
(Celentano and Wong, 1994; Jones and Westbrook, 1995; Dominguez-Perrot
et al., 1996; Haas and Macdonald, 1999). Desensitization is
generally considered a negative feedback mechanism that reduces the
peak of IPSCs, but it has been suggested recently that desensitization may, in fact, enhance GABAergic transmission by prolonging IPSCs. Jones
and Westbrook (1995) suggested that high-affinity, long-lived desensitized states delayed unbinding of GABA, thus allowing additional late openings to occur before unbinding and slowing deactivation. This
coupling of fast desensitization and deactivation provides a mechanism
that overcomes the kinetic limitations of low-affinity and low-efficacy
synaptic receptors.
Not all GABAARs, however, have the same rates of
desensitization and deactivation. We recently characterized the
distinct desensitization and deactivation kinetics of 132L and
13 GABAARs (Haas and Macdonald, 1999).
Receptors containing the 2L subunit showed prominent fast
desensitization accompanied by prolonged deactivation. In contrast, subunit-containing receptors lacked fast desensitization and
deactivated rapidly, despite having a fourfold higher GABA sensitivity.
To explore the structural bases for the coupling of fast
desensitization and deactivation, we transiently coexpressed chimeras
between the and 2L subunits and exchange mutations in these
subunits with 1 and 3 subunits in mouse L929 and human embryonic
kidney (HEK) 293T fibroblasts and recorded macroscopic
GABAAR currents evoked from excised outside-out patches using the concentration-jump technique.
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MATERIALS AND METHODS |
Construction of GABAAR chimeras
and mutations. The chimeras were constructed by using restriction
fragments (at engineered sites) or by a PCR-based overlap extension
method or by site-directed mutagenesis in existing chimeras. The
transition point of subunit amino acid sequence is listed for each
chimera, as the N-terminal parent subunit with the last amino acid of
that segment, followed by the C-terminal parent subunit with the first
amino acid of that segment: - M1e,
G232-Y235; -
M1pre-iso, Y234-T237; - M1p,
P241-C244; -
M1i, I255-N258;
- M2e,
R282-K285; -
M1e, G234-V233; and
- M1i,
I257-S256. Numbering
refers to the mature peptide. Point mutations were made using the
QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA).
Oligonucleotide primers were synthesized by the University of Michigan
DNA synthesis core facility (Ann Arbor, MI). The fidelity of the amino
acid sequences surrounding the splice sites was confirmed by sequencing of the final constructs.
Expression of recombinant GABAARs.
The cDNAs encoding rat 1, 3, 2L, and GABAAR subunit subtypes, as well as the chimeras and mutant subunits, were individually subcloned into the plasmid expression vector pCMVNeo. Mouse L929 fibroblasts (American Type Culture Collection, Rockville, MD) and HEK293T cells (a gift from P. Connely, COR Therapeutics, San Francisco, CA) were maintained in
DMEM, supplemented with 10% horse serum or 10% fetal bovine serum, respectively, at 37°C in 5% CO2 and 95% air.
Cells were transfected with 4-8 µg of each subunit plasmid along
with 1-2 µg of either EGFP plasmid for expression of the
marker green fluorescent protein (Clontech, Palo Alto, CA,) or pHOOK
(Invitrogen, Carlsbad, CA) for immunomagnetic bead separation
(Greenfield et al., 1997), using a modified calcium phosphate
coprecipitation technique as previously described (Angelotti et al.,
1993). The next day, cells were replated, and recordings were made
18-30 hr later. Expression of receptors composed of subunits
can be distinguished from expression of receptors composed of
or subunits by their smaller single-channel conductance
(Angelotti et al., 1993; Fisher and Macdonald, 1997). Preliminary
single-channel recordings were obtained for all mutant and chimeric
receptor channels, and based on their single channel conductance, each
mutant and chimera construct tested was shown to combine with 1 and
3 subtypes to produce functional receptor channels (data not shown).
Electrophysiology. Patch-clamp recordings were performed on
outside-out membrane patches excised from transfected fibroblasts bathed in an external solution consisting of (in
mM): NaCl 142; KCl or CsCl 8;
MgCl2 6; CaCl2 1; HEPES 10;
and glucose 10, pH 7.4, 325 mOsm. Glass microelectrodes were formed
from thick-walled borosilicate glass (World Precision Instruments,
Pittsburgh, PA) with a Flaming-Brown electrode puller (Sutter
Instruments, San Rafael, CA), fire-polished, then coated with Q-dope
(GC Electronics, Rockford, IL). Patch electrodes had resistances of
4-14 M
when filled with an internal solution consisting of (in
mM): KCl or CsCl 153; MgCl2
1; MgATP 2; HEPES 10; and EGTA 5, pH 7.3, 300 mOsm. This combination of
internal and external solutions produced a chloride equilibrium
potential of ~0 mV. Outside-out membrane patches were usually
voltage-clamped at 
50 to 75 mV using an EPC-7 (List, Darmstadt,
Germany) or an Axon 200A amplifier (Axon Instruments, Foster City, CA).
No voltage-dependent changes in kinetics were detected between 20 and
80 mV.
GABA was applied to outside-out membrane patches using a rapid
application system consisting of a double-barreled theta tube (Frederick Haer, Brunswick, ME) connected either to a
piezoelectric translator (Burleigh Instruments, Fishers, NY) or to a
Warner Perfusion Fast-Step (Warner Instrument Corp.ration, Hamden, CT). The fluid interface generated between control external recording solution and GABA-containing external solution was driven rapidly across the patch. The solution exchange time was monitored at the end
of each recording by blowing out the patch and stepping a dilute
external solution across the open electrode tip to measure a liquid
junction current. The 10-90% rise times for solution exchange were
consistently ~400 µsec or less with either apparatus. All
experiments were performed at room temperature (22-23°C).
For whole-cell experiments, GABA was applied to cells using multibarrel
square glass attached to the Warner Perfusion Fast-Step (Warner
Instrument Corporation). This enabled rapid solution changes, with
maximal current rise times of <10 msec. Peak GABAR currents evoked by
GABA at multiple concentrations were fitted to a sigmoidal function
using a four parameter logistic equation (sigmoidal
concentration-response) with a variable slope. The equation used to
fit the concentration-response relationship was:
where I was the peak current at a given GABA
concentration, and Imax was the
maximal peak current.
Analysis of rapid application currents. Outside-out patch
data were low-pass filtered at 2 or 3 kHz, digitized at 10 kHz, and
analyzed using the pClamp8 software suite (Axon Instruments) and Origin
4.1 (Microcal, Northampton, MA). Multiple (3-50) GABA-elicited responses were acquired for each patch at 30-60 sec intervals and
averaged to form ensemble currents for analysis. The desensitization or
deactivation time courses of ensemble GABAAR
currents were fit using the Levenberg-Marquardt least squares method
with one or two component exponential functions of the form 
an
n + C, where n is the best number of exponential
components, a is the relative amplitude of the component,
is the time constant, and C is a constant term to
account for residual current (incomplete desensitization). A second
component was accepted only if it significantly improved the fit
compared with a single exponential function, as determined by an
F test on the sum of squared residuals. Three component fits
were not considered for the application durations used in this study.
The "fast" component was defined as the faster exponential function
when the desensitization time course was fitted best by two exponential
functions. If one exponential function was sufficient, the fast phase
contribution was assigned as zero. However, to avoid misclassification,
three patches containing the
(VY
YF) mutation were
considered to have fast desensitization despite a single exponential
fit because the fast was <10 msec. Fast
desensitization was quantified as the relative contribution of the fast
exponential to the peak current, that is,
a1/(a1 + a2 + C), where
a1 and
a2 are the amplitude of the fast and
slow exponential components respectively, and C is the
constant term. The fitted peak current was given by the denominator.
Because the % fast values represent a mean of
all patches for each isoform, small values (<10%) were obtained for
isoforms in which most, but not all, patches showed no fast
desensitization (zero fast phase), not because a small fast component
was found in each patch. In none of these cases were the values
significantly different from zero. For example, only 1 of 11
patches desensitized biphasically with 26%
fast, yielding a mean % fast of 2.39% (Table
1). The extent of desensitization was
measured as (fitted peak current fitted steady-state
current)/(fitted peak current). For comparison of deactivation time
courses, a weighted summation of the fast and slow decay components
(af * f + as * s) was used, where f and s
were the fast and slow decay time constants, and
af and as
were the relative initial proportion fast and slow, respectively.
Numerical data were expressed as mean ± SEM. Statistical significance,
unless otherwise stated, was p < 0.05, using unpaired two-tailed Student's t test (with a Welch's correction for
unequal variances when necessary) or ANOVA as appropriate.
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RESULTS |
2L and subunits confer distinct desensitization and
deactivation kinetics
To evaluate the kinetics of fast desensitization, 400 msec
pulses of GABA (1 mM) were applied to excised outside-out
membrane patches containing recombinant rat
GABAAR isoforms using a rapid solution exchange
protocol (see Materials and Methods). There were no kinetic differences
observed between currents recorded from mouse (L929) and human
(HEK293T) fibroblast expression systems, and thus the results were
pooled. The current loss during the GABA pulse, attributed to receptor
desensitization, was fitted with a single or double exponential
function (see Materials and Methods). For 132L (hereafter
) receptors, rapid and extensive fast desensitization was
evident followed by a small slower phase of desensitization (Fig.
1A). The
desensitization time course was fitted best with the sum of two
exponential functions with fast (fast < 10 msec) and slow (slow ~150 msec) time
constants. The fast desensitizing component typically accounted for
>40% of the current amplitude. In contrast, for 13
(hereafter ) receptors, only minimal desensitization occurred
during the GABA application (Fig. 1B). Many
receptor currents did not desensitize at all during the 400 msec pulse.
When current loss was observed, it was fitted best with a single,
small-amplitude exponential function with a slow rate
(slow ~45 msec). Although
receptors tended to have smaller peak currents than receptors
(Haas and Macdonald, 1999; this study), there was no correlation
between peak amplitude and desensitization rate among patches of the
same isoform. The traces from Figure 1, A and B,
were rescaled and overlaid to illustrate the different desensitization
rates (Fig. 1C). Because of the presence of an additional
slow phase of desensitization for both of these isoforms ( ~1500
msec) (Haas and Macdonald, 1999), the currents did not reach steady
state during the 400 msec GABA application; however, the fast phase of
desensitization could be resolved readily.
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Figure 1.
Macroscopic kinetics of and
GABAAR isoforms. A, Rapid biphasic
desensitization and prolonged deactivation were typical for
receptors in response to a 400 msec pulse of 1 mM GABA. In
this and all subsequent figures, outside-out patches excised from
acutely transfected fibroblasts (mouse L929 and HEK293T) were exposed
to GABA using the concentration-jump technique (see Materials and
Methods). Patches were generally clamped at 50 to 75 mV. The traces
shown are the average of multiple GABA-evoked currents from a single
patch. Desensitization was described by the sum of fast
(fast) and slow (slow)
exponential functions. Deactivation was fit with one or two exponential
functions, and the weighted deactivation deact is shown.
B, Representative patch containing receptors
showed minimal desensitization during a 400 msec pulse of GABA. This
current was described by a single exponential decay function, = 34 msec. Deactivation of this isoform (deact = 54.7 ± 4.8 msec) was rapid compared with the 132L
isoform (deact = 171.8 ± 20.4 msec).
C, Overlay of rescaled traces from A and
B to emphasize the differences in fast desensitization
(p < 0.0001) and deactivation
(p < 0.0001) between these isoforms. The
current was colored gray for clarity. The
curved lines in each trace are the fitted exponential
functions describing the desensitization time course.
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The current relaxation during GABA washout (deactivation) also differed
substantially between these isoforms. Consistent with our previous
study (Haas and Macdonald, 1999), receptors deactivated slowly (deact = 171.8 ± 20.4 msec),
whereas receptors deactivated rapidly
(deact = 54.7 ± 4.8 msec) (Fig.
1A,B). The rescaled and overlaid traces also
illustrate the different deactivation rates (Fig. 1C).
Although GABA affinity can influence the time course of deactivation,
the pattern observed for these isoforms was opposite of that expected
based on EC50 alone, because receptors
have a threefold to fourfold lower GABA EC50
(Saxena and Macdonald, 1996) but deactivate rapidly compared with
receptors. Thus, minimal desensitization and rapid
deactivation were characteristic of subunit-containing receptors,
whereas pronounced fast desensitization and prolonged deactivation were
characteristic of 2L subunit-containing receptors.
Desensitization and deactivation of 13-2L
chimera receptors
We generated and 2L subunit chimeras to explore the
molecular basis for desensitization and its coupling to deactivation (Fig. 2, left). During
individual synaptic events, the duration of GABA in the cleft is brief,
and fast desensitization is the only form of desensitization involved
in shaping the synaptic current (Jones and Westbrook, 1995; Haas and
Macdonald, 1999). Therefore we were interested in evaluating
specifically the relative contribution of fast desensitization during a
400 msec pulse of saturating (1 mM) GABA (Fig. 2,
middle). Extent (percentage of current loss) and weighted
rates have been typically used to characterize desensitization, but
these measures can obscure changes specific to the physiologically
relevant fast phase. The fast phase of desensitization was defined here
as the faster exponential function when a double exponential function
was required to fit the time course of desensitization, usually
(>95%) corresponding to a fast < 15 msec
(see Materials and Methods). If a single exponential function was
sufficient to fit the current decay (usually ~30-200 msec), the
fast component amplitude was assigned as zero. Currents fitted with a
single exponential time course usually exhibited <25% current loss
during the 400 msec pulse. We summarized the desensitization patterns
as the mean relative proportion of the fast exponential component
(%fast; see Materials and Methods) (Fig. 2,
right, hatched bars). The 400 msec pulse length also allowed
deactivation to be assessed, because the extent of desensitization was
never complete with GABA applications of this duration. The deactivation time course was variable but could always be fitted with a
single or double exponential function. To facilitate comparison among
isoforms, a weighted deactivation rate (deact)
was determined (Fig. 2, right, solid bars; see Materials and
Methods).
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Figure 2.
Desensitization of
GABAARs containing -2L chimeras. Desensitization and
deactivation kinetics of GABAARs containing , 2L, or
-2L chimera subunits coexpressed with 1 and 3 subunits. The
left column shows schematics of chimeras generated
between the subunit (gray) and the 2L
subunit (white). N terminus is to the left
(N-term), and boxes (1-4)
indicate the four putative transmembrane domains. Representative
normalized responses to a 400 msec pulse of 1 mM GABA are
shown for each isoform in the center column. Desensitization was
quantified as the relative contribution of a fast phase,
%fast (hatched bars, see Materials and
Methods). Deactivation was fit with single or double exponential
functions, and for comparison the weighted sum,
deact, is shown (solid bars). Note
the different scales used for %fast and
deact in the right column. The number of
patches for each isoform is indicated in parentheses next to the sample
currents. To facilitate comparison, shaded vertical
bars indicate the mean (center of bar) and SEM range (thickness
of bar) for wild-type and values of
%fast and deact. The
asterisk indicates significant difference from both
(p < 0.01) and
(p < 0.05) receptors. Horizontal
calibration: 400 msec; vertical calibration: 6 pA for , 2 pA for
M2e, 68 pA for M1e, and 340 pA for 2L.
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The and 2L subunits share ~35% overall sequence identity,
which is much greater within the putative transmembrane domains. Therefore, splice sites for the first two - chimeras (M2e and M1e) were generated near transmembrane domains to minimize the potential for nonspecific structural perturbations in the resulting protein [see Fig. 2, left; receptors
(gray) and receptors (white)].
The M1e and M2e chimeras were designed to isolate three major
topological domains between and 2L subunits: the extracellular N
terminus; the first two transmembrane domains (TM1 and TM2), the latter
of which contains residues lining the channel pore (Xu and Akabas,
1996), and their cytoplasmic loop; and the last two transmembrane
domains (TM3 and TM4) with the large cytoplasmic loop connecting them
and the small extracellular loop between TM2 and TM3. Currents from
and receptors are shown for comparison with the
chimeras (Fig. 2, middle). Currents from receptors containing the - M2e chimera, which has subunit sequence from the N terminus through the extracellular end of TM2, were similar to
those of receptors (%fast = 2.4 ± 2.4%; deact = 54.7 ± 4.8 msec; n = 11), having no fast desensitization and rapid deactivation (M2e, %fast = 6.1 ± 4.5%,
deact = 45.4 ± 11.3 msec; n = 10) (Fig. 2, right; Table 1). Thus, subunit sequence in TM3, TM4, and the cytoplasmic loop were not
required for the subunit to abolish fast desensitization. In
contrast, the pronounced fast desensitization
(fast = 11.2 ± 2.8 msec;
%fast = 40.8 ± 6.9%; n = 14) of receptors containing the - M1e chimera was similar to
that of receptors (fast = 6.0 ± 0.7 msec; %fast = 41.3 + 3.9%;
n = 13), although the rate of deactivation was somewhat
faster (deact = 107.5 ± 14.7 msec vs
deact = 171.8 ± 20.4 msec) (Fig. 2,
right; Table 1). This chimera had subunit sequence in
the entire N terminus, yet it rapidly desensitized, indicating that the
N terminus of the subunit, potentially related to agonist binding,
was insufficient to abolish fast desensitization. The results obtained
using these chimeric subunits suggested that structures important for
fast desensitization resided within the first two transmembrane domains.
There is extensive evidence from mutation studies for a role of TM2 in
desensitization and gating in GABAARs (Im et al.,
1995; Chang et al., 1996; Dalziel et al., 2000), nicotinic
acetylcholine receptors (nAChRs) (Revah et al., 1991; Filatov
and White, 1995; Labarca et al., 1995), and
5-HT3 receptors (Yakel et al., 1993). However,
those experiments did not resolve fast phases of desensitization because of relatively slow perfusion systems, and because the implicated residues from those GABAAR studies
were identical in 2L and subunits, they could not be responsible
for the distinct differences in desensitization between receptors
containing the two subunits. The chimera strategy relies on sequence
differences, and therefore, cannot determine the importance of
conserved residues. However, given the results of using the first two
chimeras and the strong suggestion in the literature that TM2 is
involved in desensitization and gating, we mutated all four
nonidentical sites in each subunit (Fig.
3A) to the corresponding amino
acids in the other subunit to create "TM2-swap" subunits
(2L(M2S) and (M2S)) (Fig. 3B, left).
Surprisingly, neither the desensitization nor the deactivation kinetics
was sensitive to changes in TM2 sequence. The properties of receptors
containing (M2S) (%fast = 9.0 ± 4.8%; deact = 61.7 ± 12.0 msec;
n = 12) and 2L(M2S) (fast = 7.9 ± 1.3 msec; %fast = 45.4 ± 2.2%; deact = 137.8 ± 24.6 msec;
n = 9) were similar to those containing wild-type and 2L subunits, respectively (Fig. 3B, middle and
right; Table 1).
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Figure 3.
Desensitization and deactivation differences are
not specified by TM2. A, Desensitization and
deactivation kinetics of receptors containing TM2 "swapped"
subunits. The four divergent residues of TM2 shown in
(B) have been exchanged in each subunit as
indicated in the schematics. Neither desensitization nor deactivation
was altered by this exchange for either subunit. For comparison,
shaded vertical bars represent the range of values
(mean ± SEM) obtained for wild-type and
receptors. Horizontal calibration: 400 msec; vertical calibration: 10 pA for (M2S), 285 pA for 2L(M2S). B, Amino acid
sequence alignment of the second transmembrane domains of the and
2L subunits. N-terminal end (intracellular side) is to the
left. The sequence is A259 through R282; the 2L
sequence is A261 through R284. Identical residues shared by
these subunits are shaded, including the 9'
leucine (see Results).
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Three subsequent chimeras with progressively less subunit sequence
were generated, M1i, M1p, and M1pre-iso (Fig.
4B, left) to further
specify desensitization domains. A TM1 sequence alignment indicating
the splice locations is shown in Figure 4A. Although TM2 sequence did not appear to control desensitization or deactivation, several reports suggested that the nearby TM1-TM2 cytoplasmic linker
might be important. Cysteine scanning experiments suggested the channel
gate to be very close to the cytoplasm in nAChRs (Wilson and Karlin,
1998), and we previously speculated that charge differences in the
TM1-TM2 linker (NKD in the 2L subunit, SQA in the subunit) might play a role in GABAAR gating (Fisher and
Macdonald, 1997). Also, Lynch et al. (1997) showed that an alanine
substitution in the linker increased desensitization of GlyRs. The M1i
chimera contained subunit sequence through the intracellular end of TM1, whereas the TM1-TM2 linker and beyond were from the 2L
subunit. No fast desensitization, however, was observed with receptors containing this chimera (%fast = 9.6 ± 3.8%; n = 26), limiting the important structures to
those N-terminal to and including TM1. The extent of desensitization
was greater (p < 0.01) than that of
receptors (33.1 ± 5.2% compared with 12.4 ± 4.8%). Although it is possible that TM2 or the linker is involved in slower
phases of desensitization, we did not observe such effects on extent of
desensitization in the two TM2-swapped subunits or in two additional
chimeras (see below) that also contained 2L subunit sequence in
these domains.
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Figure 4.
Isolation of TM1 residues that modulate
desensitization. A, The left column shows
additional -2L chimeras used to determine the role of TM1 in
desensitization. The indicated chimeras (2L sequence is
white, subunit sequence is gray) were
expressed with 1 and 3 subunits. Representative traces are shown
in middle column. Bar graphs are as in Figure 2. Data
from the rapidly desensitizing M1e chimera (from Fig. 2) is shown again
for comparison. The asterisk indicates significant
difference from both (p < 0.01)
and (p < 0.05) receptors.
Horizontal calibration: 400 msec; vertical calibration: 4.5 pA for M1i,
22 pA for M1p, 35 pA for M1pre-iso, and 68 pA for M1e.
B, Amino acid sequence alignment of the first
transmembrane domain of the and 2L subunits. N-terminal end
(extracellular side) is to the left. The subunit
sequence is R229 through I255; the 2L subunit sequence is R231
through I257. Identical residues shared by these subunits are
shaded. The lines with
arrows indicate chimera splice positions where subunit sequence ended and 2L subunit sequence began, corresponding
to the four chimeras shown in (A). The
asterisks indicate the only residues that differ between
the M1e and M1pre-iso chimeras.
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The M1p chimera contained subunit sequence from the N terminus to
the proline in the middle of TM1, and the M1pre-iso chimera contained
subunit sequence in the N-terminus and two adjacent residues in
TM1. Both chimeras abolished fast desensitization (M1p,
%fast = 8.2 ± 8.2%, n = 6; M1pre-iso, %fast = 3.5 ± 3.5%, n = 14) (Fig. 4A, middle
and right; Table 1). These data indicated that although most
of TM1 was not involved in modulating desensitization, two TM1 residues
adjacent to the N terminus (V and Y; Fig. 4B, asterisks) were required for the subunit to abolish fast
desensitization. Consistent with the role of desensitization in shaping
deactivation patterns, the deactivation of receptors containing the
M1i, M1p, and M1pre-iso chimeras were at least as fast as
receptors (M1I, deact = 54.6 ± 8.1 msec;
M1p, deact = 10.3 ± 2.2 msec; M1pre-iso,
deact = 27.4 ± 4.9 msec). The striking
difference in fast desensitization between receptors containing the
- M1e (shown again in Fig. 4A for comparison)
and - M1pre-iso chimeras prompted us to generate exchange
mutations based on the residues that differed between these constructs.
Point mutations in TM1
The nondesensitizing receptors containing the - M1pre-iso
chimera differed from receptors with the rapidly desensitizing -
M1e chimera by only two TM1 residues, having a VY sequence in the subunit and a YF sequence in the 2L subunit (Fig. 4B, asterisks). Interestingly, the 2L subunit YF sequence is
conserved among all known , , , and GABAAR subunits across species and is similar in
the human subunit (YV); it also differs in the 
subunits, which
form nondesensitizing homomers and contain an FF sequence in that TM1
location (Amin and Weiss, 1994). Thus, we predicted that the VY
sequence in the homologous region of the subunit was responsible
for its capacity to abolish fast desensitization. We introduced these
subunit VY residues singly (2L(YV) and
2L(FY)) and together
(2L(YFVY)) into the
2L subunit to determine whether they were sufficient to attenuate
fast desensitization. However, despite their implication as the key
residues from the chimera results, the desensitization of receptors
containing these mutated 2L subunits were indistinguishable from
receptors containing the wild-type 2L subunit
(2L(YV), fast = 6.9 ± 1.4 msec;
%fast = 45.2 ± 7.8%, n = 7; 2L(FY), fast = 8.0 ± 1.7 msec,
%fast = 34.2 ± 5.2%, n = 8; 2L(YFVY), fast = 9.0 ± 1.1 msec,
%fast = 40.5 ± 5.9%, n = 19) (Fig. 5, Table 1).
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Figure 5.
VY sequence in TM1 is not sufficient to abolish
fast desensitization. The effect of mutations in TM1 of the 2L
subunit on desensitization and deactivation kinetics was determined.
The YF pair was mutated, singly and together, in the 2L subunit to
the corresponding VY residues of the subunit, and expressed with
1 and 3 subunits. The middle column illustrates
representative currents. Bar graphs are as in Figure 2. The rate and
relative contribution of fast desensitization was not altered by any of
the mutations compared to wild-type 2L subunit-containing receptors.
However, the weighted deactivation was significantly accelerated by
both the single mutations and the double mutation in TM1. The
asterisk indicates significant difference from both
(p < 0.05) and
(p < 0.01) receptors. Horizontal
calibration: 400 msec; vertical calibration: 180 pA for
2L(YV), 53 pA for
2L(FY), and 72 pA for
2L(YFVY).
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In summary, replacement of the entire N terminus of the 2L subunit
with subunit sequence (Fig. 2, - M1e chimera) had no effect
on fast desensitization, and replacement of the YF sequence in TM1 of
the 2L subunit with the corresponding VY sequence of the subunit
also failed to affect desensitization (Fig. 5,
2L(YFVY)). Fast
desensitization was abolished only when both of these exchanges were
made in the 2L subunit (Fig. 4A, -
M1pre-iso). Thus, both the N terminus and the adjacent VY residues in
TM1 of the subunit appeared to be necessary to abolish fast
desensitization. Interestingly, deactivation was substantially
accelerated when either or both of the YF pair was mutated to VY in the
2L subunit, with the fastest deactivation observed in the double
mutant (Fig. 5, 39.7 ± 4.7 msec, n = 19, compared
with 171.8 ± 20.4 msec, n = 13, with the
wild-type receptor). It appeared that prolonged deactivation had been functionally uncoupled from fast desensitization; whereas mutation of the YF sequence did not interfere with desensitization, it
prevented desensitized states from contributing to deactivation.
It is possible that pentamer assembly and/or stoichiometry differed
whether a or 2L subunit was present. Recent studies suggested
that N-terminal residues in , , and subunits were important
for intersubunit contacts related to assembly (Klausberger et al.,
2000; Taylor et al., 2000). Although subunit sequences important for
the assembly of receptors have not yet been determined, it
was possible that chimeras with subunit sequence in the N terminus
assembled differently, in terms of position or stoichiometry, than the
2L subunit mutants that contain 2L subunit N terminus sequence.
For example, if the effect of the subunit on desensitization was in
fact mediated by the VY residues but was specific to position within
the pentamer, the VY mutation in the 2L subunit might not alter
desensitization. The YF amino acid pair is conserved in all , ,
and subunits and predicted to lie near the outer vestibule of the
channel. To address the issue of position-specific effects, we studied
132L receptors containing YF to VY mutations in either or
subunits rather than in the 2L subunit. Together with the
2L(YFVY) mutation, these mutations allowed us to evaluate the impact of VY residues in TM1
at every subunit position in the GABAAR pentamer.
Neither subunit mutation abolished rapid desensitization (Fig.
6, middle and
right; Table 1), although its relative contribution was
reduced in the
1(YFVY) receptors
(%fast = 18.5 ± 5.7%,
n = 9) compared with wild type
(%fast = 41.3 ± 3.9%,
n = 13). These results were inconsistent with a simple
position-specific effect of the subunit VY sequence and provided
further evidence for the requirement of both the TM1 VY residues and
the N terminus to effectively abolish fast desensitization.
However, both subunit mutations appeared to accelerate deactivation
compared with receptors, suggesting that the uncoupling
effect observed with the
2L(YFVY) mutation
might generalize to other subunits in the pentamer
(1(YFVY), deact = 56.5 ± 8.3 msec;
3(YFVY),
deact = 99.2 ± 12.5 msec).
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Figure 6.
The effect of YF to VY mutation in the 1 or
3 subunits of receptors.
1(YFVY) was coexpressed with 3 and
2L, and 3(YFVY) was coexpressed with
1 and 2L. The middle column illustrates
representative currents. Bar graphs are as in Figure 2. The
asterisks indicates significant difference
(p < 0.05) from both and
receptors. Horizontal calibration: 400 msec; vertical
calibration: 150 pA for 1(YFVY),
and 116 pA for 3(YFVY).
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If the subunit required the combination of N-terminal
sequence and adjacent VY residues in TM1 to abolish fast
desensitization, the converse exchanges into the subunit
(introducing 2L subunit sequence) should "unmask" a rapid phase
of desensitization. Accordingly, when the VY to YF exchange was made in
the subunit, a fast component of desensitization
(fast = 9.0 ± 1.1 msec) was recorded in
13 of 21 patches (Fig. 7A,
(VYYF)). When
present, the mean contribution of this fast phase was similar to that
observed with receptors containing the 2L subunit
(%fast = 35.3 ± 3.6%,
n = 10, compared with %fast = 41.3 ± 3.9% for receptors). Although many patches did
not exhibit a fast phase, the mean rate and extent of desensitization
was significantly greater than observed with wild-type subunit
(p < 0.05). The reason for this variability in
occurrence of fast desensitization was unclear. Although mutation of
the YF residues in the 2L subunit accelerated deactivation, introduction of the YF pair into the subunit did not significantly prolong deactivation (deact = 76.5 ± 6.0 msec, compared with , deact = 54.7 ± 4.8 msec). Moreover, the deactivation rate was not
significantly different in patches having fast desensitization compared
with those that did not, suggesting that desensitized states were not
coupled to prolonged deactivation in the mutant.
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Figure 7.
2L TM1 is critical for
desensitization-deactivation coupling. A, Introducing
2L subunit sequence of extracellular TM1 or the N terminus into the
subunit increased fast desensitization (- M1e,
(VYYF)). However, prolonged
deactivation was only evident when both the N terminus and TM1
contained 2L subunit sequence (- M1i). Note that receptors
containing the (VYYF) subunit were
divided into two groups: 13 of 21 that had fast desensitization, and
eight that did not. The values for %fast were
calculated from the former group. The %fast of all 21 patches was 21.8 ± 4.4%. Splice junctions of the chimeras are
the same as the - M1e and M1i chimeras (Fig.
4A). Horizontal calibration: 400 msec; vertical
calibration: 53 pA for (VYYF), 11 pA for - M1e, and 17 pA for - M1i. B,
Representative deactivation currents were normalized to the current
amplitude at the offset of GABA application, and overlaid to illustrate
the differences in rate. Wild-type and 2L subunit-containing
receptor currents were colored gray. Note that only receptors
containing the - M1i chimera had both fast desensitization and
prolonged deactivation resembling that of receptors. Scale
bar, 100 msec.
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TM1 of the 2L subtype is critical for
desensitization-deactivation coupling
To investigate further the structures involved in fast
desensitization and its coupling to deactivation, we generated two "reverse" chimeras, splicing N-terminal 2L subunit sequence to subunit sequence (Fig. 7A, left). Whereas the initial
set of - chimeras was designed to confer a subunit-specific
property (minimal desensitization) to a 2L subunit, these -
chimeras were generated to confer properties specific to the 2L
subunit (fast desensitization and prolonged deactivation) to a subunit. We hypothesized that replacement of the subunit N terminus
by the 2L subunit N terminus, like the VY to YF exchange in ,
would confer fast desensitization. However, deactivation should remain rapid (as seen with wild-type subunit-containing receptors) because
the VY sequence would be present in TM1. We confirmed this hypothesis,
because the fast desensitization of receptors containing the -
M1e chimera resembled receptor desensitization (%fast = 40.1 ± 9.2%,
n = 7, compared with ,
%fast = 41.3 ± 3.9%), whereas the
deactivation was rapid and resembled that of receptors
(deact = 68.4 + 11.4 msec, compared
with , deact = 54.7 ± 4.8 msec)
(Fig. 7A, middle and right; Table 1). Because we were still interested in whether additional 2L subunit sequence in TM1 could restore the coupling of fast desensitization and
prolonged deactivation, we tested the - M1i chimera that extends
2L subunit sequence into TM1, including the critical YF sites (Fig.
7A, left). Consistent with the role of TM1 in
desensitization-deactivation coupling, the kinetics of both
desensitization and deactivation (Fig. 7A, middle
and right) of receptors containing this chimera were
indistinguishable from wild-type receptors
(%fast = 40.6 ± 5.7%;
deact = 186.0 ± 29.0 msec;
n = 8). The deactivation currents are expanded and
normalized in Figure 7B
TOP
ABSTRACT
INTRODUCTION
